Tobacco-derived components and materials

ABSTRACT

The invention provides a method of extracting and isolating certain compounds from tobacco. The resulting isolate can include more than 90% by weight of a given compound and can be used as a flavor component for tobacco material used in smoking articles and smokeless tobacco compositions. Exemplary compounds that may be present in the isolate according to the invention include, but are not limited to, solanone, neophytadiene, megastigmatrienone, β-damascenone, norsolanadione, cis-abienol, α-cembratrienediol, β-cembratrienediol, sucrose esters, and lutein.

FIELD OF THE INVENTION

The present invention relates to products made or derived from tobacco,or that otherwise incorporate tobacco, and are intended for humanconsumption. Of particular interest are ingredients or componentsobtained or derived from plants or portions of plants from the Nicotianaspecies.

BACKGROUND OF THE INVENTION

Popular smoking articles, such as cigarettes, have a substantiallycylindrical rod shaped structure and include a charge, roll or column ofsmokable material such as shredded tobacco (e.g., in cut filler form)surrounded by a paper wrapper thereby forming a so-called “tobacco rod.”Normally, a cigarette has a cylindrical filter element aligned in anend-to-end relationship with the tobacco rod. Typically, a filterelement comprises plasticized cellulose acetate tow circumscribed by apaper material known as “plug wrap.” Certain cigarettes incorporate afilter element having multiple segments, and one of those segments cancomprise activated charcoal particles. Typically, the filter element isattached to one end of the tobacco rod using a circumscribing wrappingmaterial known as “tipping paper.” It also has become desirable toperforate the tipping material and plug wrap, in order to providedilution of drawn mainstream smoke with ambient air. A cigarette isemployed by a smoker by lighting one end thereof and burning the tobaccorod. The smoker then receives mainstream smoke into his/her mouth bydrawing on the opposite end (e.g., the filter end) of the cigarette.

The tobacco used for cigarette manufacture is typically used in blendedform. For example, certain popular tobacco blends, commonly referred toas “American blends,” comprise mixtures of flue-cured tobacco, burleytobacco and Oriental tobacco, and in many cases, certain processedtobaccos, such as reconstituted tobacco and processed tobacco stems. Theprecise amount of each type of tobacco within a tobacco blend used forthe manufacture of a particular cigarette brand varies from brand tobrand. However, for many tobacco blends, flue-cured tobacco makes up arelatively large proportion of the blend, while Oriental tobacco makesup a relatively small proportion of the blend. See, for example, TobaccoEncyclopedia, Voges (Ed.) p. 44-45 (1984), Browne, The Design ofCigarettes, 3^(rd) Ed., p. 43 (1990) and Tobacco Production, Chemistryand Technology, Davis et al. (Eds.) p. 346 (1999).

Tobacco also may be enjoyed in a so-called “smokeless” form.Particularly popular smokeless tobacco products are employed byinserting some form of processed tobacco or tobacco-containingformulation into the mouth of the user. Various types of smokelesstobacco products are set forth in U.S. Pat. No. 1,376,586 to Schwartz;U.S. Pat. No. 3,696,917 to Levi; U.S. Pat. No. 4,513,756 to Pittman etal.; U.S. Pat. No. 4,528,993 to Sensabaugh, Jr. et al.; U.S. Pat. No.4,624,269 to Story et al.; U.S. Pat. No. 4,987,907 to Townsend; U.S.Pat. No. 5,092,352 to Sprinkle, III et al.; and U.S. Pat. No. 5,387,416to White et al.; US Pat. Appl. Pub. Nos. 2005/0244521 to Strickland etal. and 2009/0293889 to Kumar et al.; PCT WO 04/095959 to Arnarp et al.;PCT WO 05/063060 to Atchley et al.; PCT WO 05/004480 to Engstrom; PCT WO05/016036 to Bjorkholm; and PCT WO 05/041699 to Quinter et al., each ofwhich is incorporated herein by reference. See, for example, the typesof smokeless tobacco formulations, ingredients, and processingmethodologies set forth in U.S. Pat. No. 6,953,040 to Atchley et al. andU.S. Pat. No. 7,032,601 to Atchley et al., each of which is incorporatedherein by reference.

One type of smokeless tobacco product is referred to as “snuff.”Representative types of moist snuff products, commonly referred to as“snus,” have been manufactured in Europe, particularly in Sweden, by orthrough companies such as Swedish Match AB, Fiedler & Lundgren AB,Gustavus AB, Skandinavisk Tobakskompagni A/S, and Rocker Production AB.Snus products available in the U.S.A. have been marketed under thetradenames Camel Snus Frost, Camel Snus Original and Camel Snus Spice byR.J. Reynolds Tobacco Company. See also, for example, Bryzgalov et al.,1N1800 Life Cycle Assessment, Comparative Life Cycle Assessment ofGeneral Loose and Portion Snus (2005). In addition, certain qualitystandards associated with snus manufacture have been assembled as aso-called GothiaTek standard. Representative smokeless tobacco productsalso have been marketed under the tradenames Oliver Twist by House ofOliver Twist A/S; Copenhagen, Skoal, SkoalDry, Rooster, Red Seal, Husky,and Revel by U.S. Smokeless Tobacco Co.; “taboka” by Philip Morris USA;Levi Garrett, Peachy, Taylor's Pride, Kodiak, Hawken Wintergreen,Grizzly, Dental, Kentucky King, and Mammoth Cave by Conwood Company,LLC; and Camel Orbs, Camel Sticks, and Camel Strips by R.J. ReynoldsTobacco Company.

Through the years, various treatment methods and additives have beenproposed for altering the overall character or nature of tobaccomaterials utilized in tobacco products. For example, additives ortreatment processes have been utilized in order to alter the chemistryor sensory properties of the tobacco material, or in the case ofsmokable tobacco materials, to alter the chemistry or sensory propertiesof mainstream smoke generated by smoking articles including the tobaccomaterial. The sensory attributes of cigarette smoke can be enhanced byincorporating flavoring materials into various components of acigarette. Exemplary flavoring additives include menthol and products ofMaillard reactions, such as pyrazines, aminosugars, and Amadoricompounds. See also, Leffingwell et al., Tobacco Flavoring for SmokingProducts, R.J. Reynolds Tobacco Company (1972), which is incorporatedherein by reference. In some cases, treatment processes involving theuse of heat can impart to the processed tobacco a desired color orvisual character, desired sensory properties, or a desired physicalnature or texture. Various processes for preparing flavorful andaromatic compositions for use in tobacco compositions are set forth inU.S. Pat. No. 3,424,171 to Rooker; U.S. Pat. No. 3,476,118 to Luttich;U.S. Pat. No. 4,150,677 to Osborne, Jr. et al.; U.S. Pat. No. 4,986,286to Roberts et al.; U.S. Pat. No. 5,074,319 to White et al.; U.S. Pat.No. 5,099,862 to White et al.; U.S. Pat. No. 5,235,992 to Sensabaugh,Jr.; U.S. Pat. No. 5,301,694 to Raymond et al.; U.S. Pat. No. 6,298,858to Coleman, III et al.; U.S. Pat. No. 6,325,860 to Coleman, III et al.;U.S. Pat. No. 6,428,624 to Coleman, III et al.; U.S. Pat. No. 6,440,223to Dube et al.; U.S. Pat. No. 6,499,489 to Coleman, III; and U.S. Pat.No. 6,591,841 to White et al.; US Pat. Appl. Publication Nos.2004/0173228 to Coleman, III and 2010/0037903 to Coleman, III et al.,each of which is incorporated herein by reference.

The sensory attributes of smokeless tobacco can also be enhanced byincorporation of certain flavoring materials. See, for example, US Pat.Appl. Pub. Nos. 2002/0162562 to Williams; 2002/0162563 to Williams;2003/0070687 to Atchley et al.; 2004/0020503 to Williams, 2005/0178398to Breslin et al.; 2006/0191548 to Strickland et al.; 2007/0062549 toHolton, Jr. et al.; 2007/0186941 to Holton, Jr. et al.; 2007/0186942 toStrickland et al.; 2008/0029110 to Dube et al.; 2008/0029116 to Robinsonet al.; 2008/0029117 to Mua et al.; 2008/0173317 to Robinson et al.; and2008/0209586 to Neilsen et al., each of which is incorporated herein byreference.

There is a need in the art for flavorful compositions suitable foraddition to smoking products or smokeless tobacco products to introducedesired characteristics. It would be desirable to provide a method forefficient extraction and isolation of such compositions.

SUMMARY OF THE INVENTION

The present invention provides a method of extracting and isolatingvarious compounds from plants of the Nicotiana species. The method ofthe invention is selective for certain compounds that impart flavorfulcharacteristics and/or compounds that degrade to produce compounds thatimpart flavorful characteristics to smoking articles and/or smokelesstobacco products. The invention also provides methods for processingthese compounds and tobacco materials incorporating these compounds.

Thus, in one aspect, the present invention provides a method ofextracting and isolating compounds from plants of the Nicotiana species.In certain embodiments, the method comprises receiving a plant materialof the Nicotiana species; contacting the plant material with a solventfor a time and under conditions sufficient to extract one or moredesired compounds from the plant material into the solvent; separatingthe solvent containing the one or more desired compounds from theextracted plant material; and purifying the solvent containing the oneor more desired compounds to provide an isolate. In some embodiments,the isolate thus obtained comprises at least about 75 percent by weightof the one or more desired compounds. In certain embodiments, the one ormore desired compounds are selected from the group consisting ofsolanone, neophytadiene, megastigmatrienone, β-damascenone,norsolanadione, cis-abienol, α-cembratrienediol, β-cembratrienediol,sucrose esters, lutein, degradation products thereof, and mixturesthereof.

In some embodiments, the plant material of the Nicotiana species is in aform selected from the group consisting of whole leaf, laminae, cutfiller, volume expanded, stems, cut-rolled stems, cut-puffed stems,reconstituted tobacco, and particulate. In some embodiments, the plantmaterial of the Nicotiana species is provided in green form or in curedform.

In certain embodiments, the solvent is methanol. The one or more desiredcompounds thus obtained can be, for example, selected from the groupconsisting of cis-abienol, α-cembratrienediol, β-cembratrienediol,sucrose esters, and lutein. In certain embodiments, the solvent is drysteam. The one or more desired compounds thus obtained can be, forexample, selected from the group consisting of solanone, neophytadiene,megastigmatrienone, β-damascenone, and norsolanadione. When the solventis dry steam, the contacting step can further comprise collecting adistillate. In some embodiments, the distillate can comprise a waterlayer and an oily layer and the solvent containing the one or moredesired compounds can be selected from the group consisting of the oilylayer, the water layer, and a waste stream generated from thedistillation process.

In some embodiments, the purifying step comprises using preparativescale liquid chromatography. In certain embodiments, the purifying stepcomprises using flash chromatography. The purifying step can provide anisolate with a desired level of the one or more desired compounds, forexample, greater than about 90% by weight or greater than about 95% byweight of the one or more desired compounds. In certain embodiments, themethod further comprises adding the isolate to a tobacco compositionadapted for use in a smoking article or a smokeless tobacco composition.

In some embodiments, the isolate can be further treated to provide oneor more degradation products therefrom, wherein the treating comprisesoxidation (i.e., treating with H₂O₂ or another oxidizing reagent) and/orheat treatment. As one example, the isolate can comprise lutein and theone or more degradation products can be selected from the groupconsisting of megastigmatrienone, beta-damascenone, and mixturesthereof. As another example, the isolate can comprise cis-abienol andthe one or more degradation products can be selected from the groupconsisting of sclareolide, sclareol, ambroxide, and mixtures thereof.

In another aspect of the present invention is provided a method forproviding a flavor material derived from a plant of the Nicotianaspecies for addition to a tobacco composition, the method comprisingreceiving a plant material of the Nicotiana species; contacting theplant material with a solvent for a time and under conditions sufficientto extract one or more desired compounds from the plant material intothe solvent; separating the solvent containing the one or more desiredcompounds from the extracted plant material; purifying the solventcontaining the one or more compounds to provide an isolate comprising atleast about 75% by weight of the one or more desired compounds, the oneor more desired compounds being selected from the group consisting ofsolanone, neophytadiene, megastigmatrienone, β-damascenone,norsolanadione, cis-abienol, α-cembratrienediol, β-cembratrienediol,sucrose esters, lutein, degradation products thereof, and mixturesthereof; and adding the isolate to a tobacco composition adapted for usein a smoking article or a smokeless tobacco composition.

Various modifications can be made to the method of providing a flavormaterial, as noted above for the method of extracting and isolatingcompounds. For example, the plant material and solvent used can bevaried, and various additional treatment methods can be used incombinations with the inventive method.

The isolate can be added to the tobacco composition in a variety ofways. For example, the isolate can be added the form of a casingformulation or a top dressing formulation applied to tobacco strip or inthe form of a component of a reconstituted tobacco material. In certainembodiments, the tobacco composition comprises a tobacco materialadapted for use in a smoking article. In such embodiments, the amount ofisolate in the tobacco composition can be, for example, between about 5ppm and about 5 percent by weight, based on the total dry weight of thetobacco material in the smoking article. In certain embodiments, thetobacco composition comprises a tobacco material adapted for use in asmokeless tobacco product. In such embodiments, the amount of isolate inthe tobacco composition can be, for example, between about 5 ppm andabout 5 percent by weight, based on the total dry weight of the tobaccomaterial in the smokeless tobacco product.

In another aspect of the present invention is provided an isolate from aplant of the Nicotiana species or components thereof, wherein theisolate comprises at least about 75 percent by weight of a compoundselected from the group consisting of solanone, neophytadiene,megastigmatrienone, β-damascenone, norsolanadione, cis-abienol,α-cembratrienediol, β-cembratrienediol, sucrose esters, lutein, anddegradation products thereof, and mixtures thereof. In some embodiments,the isolate comprises greater than about 90% by weight or greater thanabout 95% by weight of the one or more desired compounds. In someembodiments are provided tobacco compositions comprising the isolate foruse in smoking articles or smokeless tobacco compositions.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention now will be described more fully hereinafter. Thisinvention may, however, be embodied in many different forms and shouldnot be construed as limited to the embodiments set forth herein; rather,these embodiments are provided so that this disclosure will be thoroughand complete, and will fully convey the scope of the invention to thoseskilled in the art. As used in this specification and the claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Reference to “dry weight percent” or“dry weight basis” refers to weight on the basis of dry ingredients(i.e., all ingredients except water).

The selection of the plant from the Nicotiana species can vary; and inparticular, the types of tobacco or tobaccos may vary. Tobaccos that canbe employed include flue-cured or Virginia (e.g., K326), burley,sun-cured (e.g., Indian Kurnool and Oriental tobaccos, includingKaterini, Prelip, Komotini, Xanthi and Yambol tobaccos), Maryland, dark,dark-fired, dark air cured (e.g., Passanda, Cubano, Jatin and Bezukitobaccos), light air cured (e.g., North Wisconsin and Galpao tobaccos),Indian air cured, Red Russian and Rustica tobaccos, as well as variousother rare or specialty tobaccos. Descriptions of various types oftobaccos, growing practices and harvesting practices are set forth inTobacco Production, Chemistry and Technology, Davis et al. (Eds.)(1999), which is incorporated herein by reference. Variousrepresentative types of plants from the Nicotiana species are set forthin Goodspeed, The Genus Nicotiana, (Chonica Botanica) (1954); U.S. Pat.No. 4,660,577 to Sensabaugh, Jr. et al.; U.S. Pat. No. 5,387,416 toWhite et al. and U.S. Pat. No. 7,025,066 to Lawson et al.; US PatentAppl. Pub. Nos. 2006/0037623 to Lawrence, Jr. and 2008/0245377 toMarshall et al.; each of which is incorporated herein by reference.Exemplary Nicotiana species include are N. tabacum, N. rustica, N.alata, N. arentsii, N. excelsior, N. forgetiana, N. glauca, N.glutinosa, N. gossei, N. kawakamii, N. knightiana, N. langsdorffi, N.otophora, N. setchelli, N. sylvestris, N. tomentosa, N. tomentosiformis,N. undulata, N. x sanderae, N. africana, N. amplexicaulis, N.benavidesii, N. bonariensis, N. debneyi, N. longiflora, N. maritina, N.megalosiphon, N. occidentalis, N. paniculata, N. plumbaginifolia, N.raimondii, N. rosulata, N. simulans, N. stocktonii, N. suaveolens, N.umbratica, N. velutina, N. wigandioides, N. acaulis, N. acuminata, N.attenuata, N. benthamiana, N. cavicola, N. clevelandii, N. cordifolia,N. corymbosa, N. fragrans, N. goodspeedii, N. linearis, N. miersii, N.nudicaulis, N. obtusifolia, N. occidentalis subsp. Hersperis, N.pauciflora, N. petunioides, N. quadrivalvis, N. repanda, N.rotundifolia, N. solanifolia and N. spegazzinii.

Nicotiana species can be derived using genetic-modification orcrossbreeding techniques (e.g., tobacco plants can be geneticallyengineered or crossbred to increase or decrease production ofcomponents, characteristics or attributes). See, for example, the typesof genetic modifications of plants set forth in U.S. Pat. No. 5,539,093to Fitzmaurice et al.; U.S. Pat. No. 5,668,295 to Wahab et al.; U.S.Pat. No. 5,705,624 to Fitzmaurice et al.; U.S. Pat. No. 5,844,119 toWeigl; U.S. Pat. No. 6,730,832 to Dominguez et al.; U.S. Pat. No.7,173,170 to Liu et al.; U.S. Pat. No. 7,208,659 to Colliver et al. andU.S. Pat. No. 7,230,160 to Benning et al.; US Patent Appl. Pub. No.2006/0236434 to Conkling et al.; and PCT WO2008/103935 to Nielsen et al.

For the preparation of smokeless and smokable tobacco products, it istypical for harvested plants of the Nicotiana species to be subjected toa curing process. Descriptions of various types of curing processes forvarious types of tobaccos are set forth in Tobacco Production, Chemistryand Technology, Davis et al. (Eds.) (1999). Exemplary techniques andconditions for curing flue-cured tobacco are set forth in Nestor et al.,Beitrage Tabakforsch. Int., 20, 467-475 (2003) and U.S. Pat. No.6,895,974 to Peele, which are incorporated herein by reference.Representative techniques and conditions for air curing tobacco are setforth in Roton et al., Beitrage Tabakforsch. Int., 21, 305-320 (2005)and Staaf et al., Beitrage Tabakforsch. Int., 21, 321-330 (2005), whichare incorporated herein by reference. Certain types of tobaccos can besubjected to alternative types of curing processes, such as fire curingor sun curing. Preferably, harvested tobaccos that are cured are thenaged.

At least a portion of the plant of the Nicotiana species (e.g., at leasta portion of the tobacco portion) can be employed in an immature form.That is, the plant, or at least one portion of that plant, can beharvested before reaching a stage normally regarded as ripe or mature.As such, for example, tobacco can be harvested when the tobacco plant isat the point of a sprout, is commencing leaf formation, is commencingflowering, or the like. At least a portion of the plant of the Nicotianaspecies (e.g., at least a portion of the tobacco portion) can beemployed in a mature form. That is, the plant, or at least one portionof that plant, can be harvested when that plant (or plant portion)reaches a point that is traditionally viewed as being ripe, over-ripe ormature. As such, for example, through the use of tobacco harvestingtechniques conventionally employed by farmers, Oriental tobacco plantscan be harvested, burley tobacco plants can be harvested, or Virginiatobacco leaves can be harvested or primed by stalk position.

In accordance with the present invention, a tobacco product incorporatestobacco that is combined with one or more compounds extracted and/orisolated from a plant of the Nicotiana species in green form or curedform. At least a portion of the tobacco product can comprise compoundsremoved from the Nicotiana plant (e.g., by extraction, distillation, orother types of processing techniques). In some embodiments, at least aportion of the tobacco product can be composed of degradation productsderived from these compounds, such as compounds collected aftersubjecting the plants to chemical reaction or after subjecting compoundsor mixtures thereof isolated from Nicotiana plants to chemical reaction(e.g., acid/base reaction conditions, oxidation conditions, enzymatictreatment, and/or heat treatment).

The Nicotiana species can be selected for the content of variouscompounds that are present therein. For example, plants can be selectedon the basis that those plants produce relatively high quantities of oneor more of the compounds desired to be isolated therefrom. In certainembodiments, plants of the Nicotiana species (e.g., Galpao communtobacco) are specifically grown for their abundance of leaf surfacecompounds. Tobacco plants can be grown in greenhouses, growth chambers,or outdoors in fields, or grown hydroponically.

Various parts or portions of the plant of the Nicotiana species can beemployed. For example, virtually all of the plant (e.g., the wholeplant) can be harvested, and employed as such. Alternatively, variousparts or pieces of the plant can be harvested or separated for furtheruse after harvest. For example, the leaves, stem, stalk, and variouscombinations thereof, can be isolated for further use or treatment. Theplant material of the invention may thus comprise an entire plant or anyportion of a plant of the Nicotiana species.

The post-harvest processing of the plant or portion thereof can vary.After harvest, the plant or portion thereof can be used in a green form(e.g., the plant or portion thereof can be used without being subjectedto any curing process). For example, the plant or portion thereof can beused without being subjected to significant storage, handling orprocessing conditions. In certain situations, it is preferable that theplant or portion thereof be used virtually immediately after harvest.Alternatively, for example, a plant or portion thereof in green form canbe refrigerated or frozen for later use, freeze dried, subjected toirradiation, yellowed, dried, cured (e.g., using air drying techniquesor techniques that employ application of heat), heated or cooked (e.g.,roasted, fried or boiled), or otherwise subjected to storage ortreatment for later use.

The harvested plant or portion thereof can be physically processed. Theplant or portion thereof can be separated into individual parts orpieces (e.g., the leaves can be removed from the stems, and/or the stemsand leaves can be removed from the stalk. The harvested plant orindividual parts or pieces can be further subdivided into parts orpieces (e.g., the leaves can be shredded, cut, comminuted, pulverized,milled or ground into pieces or parts that can be characterized asfiller-type pieces, granules, particulates or fine powders). The plant,or parts thereof, can be subjected to external forces or pressure (e.g.,by being pressed or subjected to roll treatment). When carrying out suchprocessing conditions, the plant or portion thereof can have a moisturecontent that approximates its natural moisture content (e.g., itsmoisture content immediately upon harvest), a moisture content achievedby adding moisture to the plant or portion thereof, or a moisturecontent that results from the drying of the plant or portion thereof.For example, powdered, pulverized, ground or milled pieces of plants orportions thereof can have moisture contents of less than about 25 weightpercent, often less than about 20 weight percent, and frequently lessthan about 15 weight percent.

The plant of the Nicotiana species or portions thereof can be subjectedto other types of processing conditions. For example, components can beseparated from one another or otherwise fractionated into chemicalclasses or mixtures of individual compounds. Typical separationprocesses can include one or more process steps (e.g., solventextraction using polar solvents, organic solvents, or supercriticalfluids), chromatography, distillation, filtration, recrystallization,and/or solvent-solvent partitioning. Exemplary extraction and separationsolvents or carriers include water, alcohols (e.g., methanol orethanol), hydrocarbons (e.g., heptane and hexane), diethyl ether,methylene chloride and supercritical carbon dioxide. Exemplarytechniques useful for extracting components from Nicotiana species aredescribed in U.S. Pat. No. 4,144,895 to Fiore; U.S. Pat. No. 4,150,677to Osborne, Jr. et al.; U.S. Pat. No. 4,267,847 to Reid; U.S. Pat. No.4,289,147 to Wildman et al.; U.S. Pat. No. 4,351,346 to Brummer et al.;U.S. Pat. No. 4,359,059 to Brummer et al.; U.S. Pat. No. 4,506,682 toMuller; U.S. Pat. No. 4,589,428 to Keritsis; U.S. Pat. No. 4,605,016 toSoga et al.; U.S. Pat. No. 4,716,911 to Poulose et al.; U.S. Pat. No.4,727,889 to Niven, Jr. et al.; U.S. Pat. No. 4,887,618 to Bernasek etal.; U.S. Pat. No. 4,941,484 to Clapp et al.; U.S. Pat. No. 4,967,771 toFagg et al.; U.S. Pat. No. 4,986,286 to Roberts et al.; U.S. Pat. No.5,005,593 to Fagg et al.; U.S. Pat. No. 5,018,540 to Grubbs et al.; U.S.Pat. No. 5,060,669 to White et al.; U.S. Pat. No. 5,065,775 to Fagg;U.S. Pat. No. 5,074,319 to White et al.; U.S. Pat. No. 5,099,862 toWhite et al.; U.S. Pat. No. 5,121,757 to White et al.; U.S. Pat. No.5,131,414 to Fagg; U.S. Pat. No. 5,131,415 to Munoz et al.; U.S. Pat.No. 5,148,819 to Fagg; U.S. Pat. No. 5,197,494 to Kramer; U.S. Pat. No.5,230,354 to Smith et al.; U.S. Pat. No. 5,234,008 to Fagg; U.S. Pat.No. 5,243,999 to Smith; U.S. Pat. No. 5,301,694 to Raymond et al.; U.S.Pat. No. 5,318,050 to Gonzalez-Parra et al.; U.S. Pat. No. 5,343,879 toTeague; U.S. Pat. No. 5,360,022 to Newton; U.S. Pat. No. 5,435,325 toClapp et al.; U.S. Pat. No. 5,445,169 to Brinkley et al.; U.S. Pat. No.6,131,584 to Lauterbach; U.S. Pat. No. 6,298,859 to Kierulff et al.;U.S. Pat. No. 6,772,767 to Mua et al.; and U.S. Pat. No. 7,337,782 toThompson, all of which are incorporated herein by reference. See also,the types of separation techniques set forth in Brandt et al., LC-GCEurope, p. 2-5 (March, 2002) and Wellings, A Practical Handbook ofPreparative HPLC (2006), which are incorporated herein by reference. Inaddition, the plant or portions thereof can be subjected to the types oftreatments set forth in Ishikawa et al., Chem. Pharm. Bull., 50, 501-507(2002); Tienpont et al., Anal. Bioanal. Chem., 373, 46-55 (2002);Ochiai, Gerstel Solutions Worldwide, 6, 17-19 (2006); Coleman, III, etal., J. Sci. Food and Agric., 84, 1223-1228 (2004); Coleman, III et al.,J. Sci. Food and Agric., 85, 2645-2654 (2005); Pawliszyn, ed.,Applications of Solid Phase Microextraction, RSC ChromatographyMonographs, (Royal Society of Chemistry, UK) (1999); Sahraoui et al., J.Chrom., 1210, 229-233 (2008); and U.S. Pat. No. 5,301,694 to Raymond etal., which are incorporated herein by reference.

In particular, in certain embodiments, one or more compounds of interestare extracted from a plant material of the Nicotiana species or aportion thereof by contacting the plant or portion thereof with asolvent under conditions (e.g., suitable pressure and temperature)sufficient to extract one or more desired compounds from the plantmaterial. In some embodiments, the solvent is an organic solvent, suchas methanol or hexanes. In other embodiments, the solvent is dry steam.Dry steam (also referred to as anhydrous steam) is steam having aminimal content of suspended water particles (i.e., moisture). Forexample, dry steam typically comprises less than about 5% waterparticles by weight or less than about 10% water particles by weight.

The temperature and pressure at which the extraction process isconducted can vary. However, in some embodiments, suitable extraction isachieved at ambient temperature and pressure. Further, the amount oftime that the solvent is in contact with the tobacco material can vary.Typically, the solvent will remain in contact with the tobacco materialfor approximately two hours, although longer or shorter time periods canbe used without departing from the invention.

Exemplary compounds of interest that can be present in the extractobtained according to the methods of the present invention include, butare not limited to, solanone, neophytadiene, megastigmatrienone,β-damascenone, norsolanadione, cis-abienol, α-cembratrienediol,β-cembratrienediol, sucrose esters, and/or lutein.

Further processing of the extracted product can be carried out in anumber of ways. The method of further processing can depend on thecompounds present in the extract and/or the type of solvent used in theextraction.

For example, where an organic solvent (e.g., methanol or hexanes) isused to extract one or more compounds from the tobacco material, thesolvent brought into contact with the tobacco material can simply befiltered to remove particulate tobacco material and the filtrate can beconcentrated.

Where dry steam is used to extract one or more compounds from thetobacco material, the dry steam is typically condensed following thecontacting step to give a steam distillate. In certain embodiments, thisdistillation method is conducted on various mixtures of cured tobaccomaterials. For example, in one embodiment, a mixture of flue-cured,burley, and Oriental tobaccos is used. Methods used for the productionof essential oils can be used herein to distill compounds of interestfrom tobacco plants or portions thereof. For exemplary steamdistillation processes and conditions that can be used or modified foruse to provide compounds of interest from tobacco plants or portionsthereof according to the present invention, see for example, U.S. Pat.No. 5,891,501 to McKellip et al., which is incorporated herein byreference. The type of apparatus used to process the material in thisway can be, for example, the type traditionally employed for theisolation of peppermint oils. In certain embodiments, the steamdistillate comprises a water condensate layer and an oily layer that canbe separated from each other.

The oily layer thus obtained from the tobacco material typicallycomprises one or more compounds of interest (e.g., neophytadiene,solanone, megastigmatrienone isomers, β-damascenone, andnorsolanadione). In certain embodiments, the oily layer advantageouslyis rich in flavor compounds, including one or more of the compounds ofinterest noted above, and is essentially alkaloid free. For example, incertain embodiments, the oily layer contains less than about 15% byweight, less than about 10% by weight, less than about 5% by weight,less than about 2% by weight, less than about 1% by weight, or less thanabout 0.5% by weight alkaloids. The percentages of other compoundspresent in the oily layer can vary, depending for example, on the typeof tobacco subjected to distillation by the methods provided herein.

The waste water resulting from the distillation process may, in certainembodiments, also comprise compounds of interest (e.g., including, butnot limited to, those compounds of interest noted to be present in theoily layer). These compounds are believed to be present both in the oilylayer and in the water because the distillation process can providecertain compounds with notable solubility in water. Therefore, in someembodiments, compounds of interest are isolated from the waste waterproduced by the distillation process. For example, in some embodiments,flavor compounds and/or nicotine are present in the waste water. It isnoted that the percentage of various volatile and semi-volatilecompounds in the waste water varies as a function of time. Thus, variousfractions of the waste water can be collected separately to providesolutions containing higher percentages of certain compounds.Accordingly, the invention provides for the extraction of desiredcompounds by distillation, wherein the desired compounds are provided inthe oily layer of the distillate, the water layer of the distillate,and/or in the waste water produced during the distillation process.

Various compounds or mixtures of compounds from the Nicotiana plant orportions thereof can be isolated by the methods provided herein. As usedherein, an “isolated component,” or “plant isolate,” is a compound orcomplex mixture of compounds separated from a plant of the Nicotianaspecies or a portion thereof. The isolated component can be a singlecompound, a homologous mixture of similar compounds (e.g., isomers of aflavor compound), or a heterologous mixture of dissimilar compounds(e.g., a complex mixture of various compounds of different types,preferably having desirable sensory attributes). Tobacco material thathas been subjected to the extraction methods described herein may befurther processed, e.g., to extract one or more additional compoundstherefrom. See, for example, US Patent App. Publ. No. 2008/0254149 toHavkin-Frenkel, which is incorporated herein by reference.

According to the present invention, a variety of compounds havingdistinctive flavor and aroma characteristics can be extracted and/orisolated from plants of the Nicotiana species. Certain of thosecompounds can be considered to be volatile under normal ambientconditions of temperature, humidity and air pressure. Preferredcompounds exhibit positive sensory attributes at relatively lowconcentrations. Examples of the types of compounds that can be presentin Nicotiana plants and extracted and isolated by the methods describedherein include solanone, neophytadiene, megastigmatrienone,β-damascenone, norsolanadione, cis-abienol, α-cembratrienediol,β-cembratrienediol, sucrose esters, and/or lutein.

Cis-abienol is a major labdanoid in the green leaf of tobaccos. Forexample, cis-abienol is commonly found in Oriental tobaccos.

During air- and sun-curing of green Oriental tobaccos, the concentrationof cis-abienol typically decreases significantly, as numerous labdanoiddegradation products are formed. The labdanoid degradation products arestructurally similar to cis-abienol, and include, for example,sclareolide, sclareol, and ambroxide. These degradation products areknown to impart cedar characteristics to tobacco products containingOriental tobaccos.

Isolated cis-abienol may find a number of applications in smokingproducts and/or smokeless tobacco products. In some embodiments,isolated cis-abienol is degraded by various means and the degradationproducts can be used in smoking products and/or smokeless tobaccoproducts. For example, in certain embodiments, isolated cis-abienoland/or degradation products thereof find use as tobacco-derived, naturalflavor materials (e.g., Turkish replacement flavor material) or Orientaltobacco replacements. In some embodiments, cis-abienol and/ordegradation products are used for therapeutic or neutriceuticalapplications. For example, labdanoid compounds have shown anti-canceractivity. See, for example, Jung et al., Bioorg. Med. Chem. Lett. 8:3295-98 (1998), which is incorporated herein by reference. In someembodiments, cis-abienol is used as a substrate in the preparation ofother compounds. For example, it has been used as a substrate in thesynthesis of weidendiols, which are cholesterol ester transfer proteininhibitors that may reduce the risk of atherosclerosis. See, forexample, Barrero et al., Tetrahedron 54: 5635-5650 (1998), which isincorporated herein by reference.

Cembratrienediols (e.g., α-2,7,11-cembratriene-4,6-diol andβ-2,7,11-cembratriene-4,6-diol) are found in high quantities in the leafand flower of Nicotiana tabacum. Biodegradation of these compoundsduring curing of tobacco leaves results in the formation of a range offlavor compounds. Cembratrienediols have a structure as depicted below.

Isolated cembratrienediols may find a number of applications in smokingproducts and/or smokeless tobacco products. In some embodiments,isolated cembratriene diols are degraded by various means and thedegradation products are used in smoking products and/or smokelesstobacco products. For example, in certain embodiments, isolatedcembratriene diols and/or degradation products thereof may find use astobacco-derived, natural flavor materials. In some embodiments,cembratriene diols and/or degradation products are used for therapeuticapplications. For example, cembratriene diols have been found to providepotential health benefits in the treatment of cancer and certainneurodegenerative diseases. See, for example, Saito et al., Agric. Biol.Chem. 51(3): 941-43 (1987) and U.S. Pat. No. 4,701,570 to Mizusaki etal., which are incorporated herein by reference. Cembratriene diols andderivatives thereof may also be useful as drugs to prevent smokingand/or to treat nicotine addiction. See, for example, El Sayed et al.,Expert Opin. Invest. Drugs 16(6): 877-87 (2007).

Sucrose esters are glycolipid compounds, characterized by low molecularweight carboxylic acids attached to hydroxide groups of the glucoseportion of sucrose. Sucrose esters are considered to be some of the mostimportant aroma and sensory precursors responsible for Oriental tobaccoflavor. See, for example, Leffingwell et al., Rec. Adv. Tob. Sci. 14:169-218 (1998), which is incorporated herein by reference.

Sucrose esters are typically sucrose molecules comprising three acylgroups on the glucose ring, each hydrocarbon chain of the acyl groupcomprising 3-8 carbon atoms and optionally including one or more doublebonds. The sucrose esters also typically include an acetyl group oneither the glucose ring or the fructose ring, which gives rise to thecommon reference to these esters as tetra-acyl sucrose esters. The exactstructure of the sucrose esters isolated according to the presentinvention can vary somewhat as to placement, chain length, andsaturation of the acyl groups, most sucrose esters of Oriental tobaccoare encompassed by the following structure:

wherein each R is an independently selected C3-C8 hydrocarbon, which canbe straight or branched and saturated or unsaturated, and both R₁substituents are H or one R₁ is H and the other is acetyl (—C(O)CH₃).Most common R groups comprise butyl, pentyl, and hexyl.

Lutein is a major carotenoid pigment in tobacco.

Lutein is known to break down during the air-curing of green tobacco toproduce several ionones and derivatives thereof. Two important types ofderivatives of lutein are megastigmatrienones and beta-damascenone.These compounds impact the aroma characteristics of cured tobacco. Inaddition to being degradation products of lutein, megastigmatrienone andbeta-damascenone may also be separately extracted and isolated fromtobacco according to the methods provided herein.

Isolated lutein may find a number of applications in smoking productsand/or smokeless tobacco products. In some embodiments, isolated luteinis degraded by various means and the degradation products producedtherefrom can be used in smoking products and/or smokeless tobaccoproducts. For example, in certain embodiments, isolated lutein and/ordegradation products thereof may find use as tobacco-derived, naturalflavor materials, colorants, or antioxidants. Lutein is also useful as anutritional and/or therapeutic compound. See for example, Granado etal., Br. J. Nutr. 90(3): 487-502 (2003); Sies et al., Int. J. Vitam.Nutr. Res. 73(2): 95-100 (2003); and Krinsky et al., Annu. Rev. Nutr.23(2): 171-201 (2003), which are incorporated herein by reference.

Solanone is a compound that is a useful tobacco flavorant and flavorenhancer. It is specifically noted to be present in burley tobaccoaroma. See, for example, Domle et al., Helv. Chim. Acta, 55(6):1866-1882 (1972), which is incorporated herein by reference. Although itis commonly produced by synthetic means (see, e.g., Johnson et al., J.Org. Chem. 30(9): 2918-2921 (1965); and U.S. Pat. No. 4,433,695 to Hallet al. and U.S. Pat. No. 4,547,594 to Light et al., which areincorporated herein by reference), solanone is present in tobacco andmay be isolated according to the methods provided herein.

Isolated solanone may find a number of applications in smoking productsand/or smokeless tobacco products. In some embodiments, isolatedsolanone may be degraded by various means and the degradation productsmay be used in smoking products and/or smokeless tobacco products. Forexample, in certain embodiments, isolated solanone and/or degradationproducts thereof may find use as tobacco-derived, natural flavormaterials.

Neophytadiene is reported to be a tobacco flavor enhancer in that it mayact as a flavor carrier by entrapping volatiles in the tobacco smokeaerosol. See, for example, J. C. Leffingwell, Leaf chemistry: basicchemical constituents of tobacco leaf and differences among tobaccotypes. Tobacco: Production, Chemistry and Technology, 265-284 (1999).

Isolated neophytadiene may find a number of applications in smokingproducts and/or smokeless tobacco products. In some embodiments,isolated neophytadiene may be degraded by various means and thedegradation products may be used in smoking products and/or smokelesstobacco products. For example, in certain embodiments, isolatedneophytadiene and/or degradation products thereof may find use astobacco-derived, natural flavor materials.

Norsolanadione is another compound that is known to be useful as atobacco flavorant and in augmenting or enhancing the aroma and taste ofsmoking tobacco. Like solanone, this compound is commonly synthesized,rather than isolated. See, for example, U.S. Pat. No. 4,517,385 to Lightet al., which is incorporated herein by reference.

Isolated norsolanadione may find a number of applications in smokingproducts and/or smokeless tobacco products. In some embodiments,isolated norsolanadione may be degraded by various means and thedegradation products may be used in tobacco products. For example, incertain embodiments, isolated norsolanadione and/or degradation productsthereof may find use as tobacco-derived, natural flavor materials.

Following extraction of compounds of interest from tobacco, it isdesirable according to the present invention to further isolate andpurify certain compounds. Because the extraction processes disclosedherein typically lead to complex mixtures of compounds, they must befurther treated to provide desired mixtures of compounds and/or singleisolated compounds (e.g., to give isolates comprising at least about 75%by weight of the compound or compounds).

The means by which such mixtures and/or single isolated compounds areprovided can vary. Additional solvent extractions (e.g., solventextraction using polar solvents, organic solvents, or supercriticalfluids), chromatography, distillation, filtration, recrystallization,and/or solvent-solvent partitioning may be used to isolate and/or purifydesired compounds from the extracts.

In some embodiments, multiple methods are used to isolate and/or purifythe desired compounds. For example, solvent extraction may be combinedwith one or more chromatographic methods. The sample obtained viaextraction may be dissolved in a solvent and injected directly onto theflash chromatography system or may be treated in some way prior toinjection. In another example, in some embodiments, the sample is firsttreated to remove one or more compounds that are known to elute undersimilar conditions as the compound(s) to be isolated by flashchromatography. In one particular embodiment, an extract obtained bymethanol extraction of a tobacco material is processed to removequercetin-3-rutinoside (“rutin”). For example, the rutin can be removedfrom the extract by adding water, methanol, and methylene chloride tothe extract and extracting the rutin into the methanol/water layer. Themethylene chloride layer can be concentrated and further processed(e.g., by chromatography) to isolate and/or purify the desiredcompound(s) contained therein. In other embodiments, the extract can bedissolved and directly subjected to chromatographic separation.

In some embodiments, preparative liquid chromatography is used toisolate and/or purify certain compounds of interest from a tobaccoextract. In some embodiments, a compound or compounds of interest areisolated using preparative liquid chromatography based on the elutiontimes of standards. Various automated commercial prep-LC systems areavailable, from manufacturers including Waters, Agilent Technologies,and Bio-Rad. The specific parameters of the prep LC system used can bevaried by one of skill in the art to achieve the desired level ofresolution. For example, the solvent may be any solvent or mixture ofsolvents sufficient to dissolve the compound(s) of interest. The solventmay be, for example, water, methanol, ethanol, ethyl acetate, diethylether, methylene chloride, chloroform, petroleum ether, and/or hexanes.The system may be operated with an isocratic or gradient solvent system(i.e., varying the ratio of two or more solvents as a function of time).In some embodiments, the solvent system can be chosen such that itprovides the best resolution between the compound of interest and othercompounds present in the mixture. The flow rate of the system may bevaried, for example, from about 10 mL/min to about 100 mL/min (e.g.,about 36 mL/min).

In some embodiments, flash chromatography is used to isolate and/orpurify certain compounds of interest from a tobacco extract. Flashchromatography systems are known in the art and exemplary systems arediscussed, for example, in Still et al., J. Org. Chem. 42: 2923-2925(1978) and U.S. Pat. No. 4,591,442 to Andrews, which are incorporatedherein by reference. Various automated commercial flash chromatographysystems are available, from manufacturers including Biotage, TeledyneIsco, Grace Davison Discovery Sciences, and Buchi. Flash chromatographymay be desirable to provide reasonably large quantities of compound, ascolumns typically have relatively large particle sizes (e.g., roughly30-40 μm) and can accommodate a greater quantity of sample (and a moreconcentrated sample), allowing more of the compound(s) of interest to beisolated per injection.

The specific parameters of the flash chromatography system used can bevaried by one of skill in the art to achieve the desired level ofresolution. For example, the solvent may be any solvent or mixture ofsolvents sufficient to dissolve the compound(s) of interest. The solventmay be, for example, water, methanol, ethanol, ethyl acetate, diethylether, methylene chloride, chloroform, petroleum ether, and/or hexanes.The system may be operated with an isocratic or gradient solvent system(i.e., varying the ratio of two or more solvents as a function of time).In some embodiments, the solvent system may be chosen to provide thebest resolution between the compound of interest and other compoundspresent in the mixture. The flow rate of the system may be varied, forexample, from about 20 to about 200 mL/min (e.g., about 150 mL/min).

Flash chromatography may or may not provide the compound(s) of interestat a sufficient purity level. In certain embodiments, the fractionscorresponding to the compound(s) of interest may be collected, combined,and concentrated to give an isolate comprising the compound(s) ofinterest at a sufficient level of purity (i.e., wherein the compound(s)of interest are present in a sufficient weight percentage of theisolate). For example, the isolate of the present invention can comprisethe compound(s) of interest in an amount of greater than about 75% byweight, greater than about 80% by weight, greater than about 85% byweight, greater than about 90% by weight, greater than about 95% byweight, greater than about 98% by weight, or greater than about 99% byweight. In some embodiments, fractions obtained from flashchromatography can be further resolved using preparative liquidchromatography.

In some embodiments, isolated compounds or mixtures thereof can besubjected to conditions so as to cause those compounds to undergochemical transformation. For example, the tobacco material obtained fromplants of the Nicotiana species or portion thereof can be treated tocause chemical transformation or be admixed with other ingredients. Insome embodiments, the extracts obtained therefrom, or the isolatedcompound(s) (isolates) can be treated to cause chemical transformationor be admixed with other ingredients. The chemical transformations ormodification of the tobacco material, extract, or isolated compound canresult in changes of certain chemical and physical properties of thetobacco material, extract, or isolated compound(s) (e.g., the sensoryattributes thereof). Exemplary chemical modification processes can becarried out by acid/base reaction, hydrolysis, oxidation, heating and/orenzymatic treatments; and as such, compounds can undergo variousdegradation reactions.

In certain embodiments, the tobacco material, extract, or isolate istreated to provide degradation products (e.g., lutein may be treated toprovide various flavor compounds, including megastigmatrienones and/orβ-damascenone; cis-abienol may be treated to provide sclareolide,sclareol, and/or ambroxide). Degradation products are any compounds thatare produced from the compounds extracted and/or isolated according tothe present invention. Degradation products can be formed naturally fromsuch compounds or may be produced by an accelerated degradation process(e.g., by the addition of heat and/or chemicals to accelerate thebreakdown of the compounds). These compounds can be degraded, forexample, by means of oxidation (e.g., through treatment with H₂O₂ orother oxidizing agents) and/or hydrolysis reactions.

Exemplary types of further ingredients that can be admixed with thetobacco material, extracts, or isolates according to the presentinvention include one or more flavorants, fillers, binders, pHadjusters, buffering agents, colorants, disintegration aids,antioxidants, humectants and preservatives.

The extracts and isolates of the present invention are useful ascomponents added to tobacco compositions, particularly tobaccocompositions incorporated into smoking articles or smokeless tobaccoproducts. Addition of the extracts or isolates to a tobacco compositioncan enhance the tobacco composition in a variety of ways, depending onthe nature of the extract or isolate and the type of tobaccocomposition. Exemplary extracts and isolates can serve to provide flavorand/or aroma to a tobacco product (e.g., composition that alters thesensory characteristics of tobacco compositions or smoke derivedtherefrom). The extracts and isolates of the invention can also be usedas components of tobacco products that contain no other tobacco materialtherein. In other words, the extract or isolate of the invention couldbe used as the sole source of tobacco in the tobacco product of theinvention by, for example, incorporating the extract or isolate into anoral smokeless tobacco composition, such as a product adapted todissolve or melt in the oral cavity.

The form of the extract or isolate obtained according to the presentinvention can vary. Typically, the isolate is in a solid, liquid, orsemi-solid or gel form. The isolate can be used in concrete, absolute,or neat form. Solid forms of the isolate include spray-dried andfreeze-dried forms. Liquid forms of the isolate include isolatecontained within aqueous or organic solvent carriers.

The extract or isolate can be employed as a component of a tobaccocomposition in a variety of ways. The extract or isolate can be employedas a component of processed tobaccos. In one regard, the extract orisolate can be employed within a casing formulation for application totobacco strip (e.g., using the types of manners and methods set forth inU.S. Pat. No. 4,819,668 to Shelar, which is incorporated herein byreference) or within a top dressing formulation. Alternatively, theextract or isolate can be employed as an ingredient of a reconstitutedtobacco material (e.g., using the types of tobacco reconstitutionprocesses generally set forth in U.S. Pat. No. 5,143,097 to Sohn; U.S.Pat. No. 5,159,942 to Brinkley et al.; U.S. Pat. No. 5,598,868 to Jakob;U.S. Pat. No. 5,715,844 to Young; U.S. Pat. No. 5,724,998 to Gellatly;and U.S. Pat. No. 6,216,706 to Kumar, which are incorporated herein byreference). The extract or isolate also can be incorporated into acigarette filter (e.g., in the filter plug, plug wrap, or tipping paper)or incorporated into cigarette wrapping paper, preferably on the insidesurface, during the cigarette manufacturing process.

The Nicotiana-derived extract or isolate can be incorporated intosmoking articles. Representative tobacco blends, non-tobacco components,and representative cigarettes manufactured therefrom, are set forth inU.S. Pat. No. 4,836,224 to Lawson et al.; U.S. Pat. No. 4,924,888 toPerfetti et al.; U.S. Pat. No. 5,056,537 to Brown et al.; U.S. Pat. No.5,220,930 to Gentry; and U.S. Pat. No. 5,360,023 to Blakley et al.; USPat. Application 2002/0000235 to Shafer et al.; and PCT WO 02/37990.Those tobacco materials also can be employed for the manufacture ofthose types of cigarettes that are described in U.S. Pat. No. 4,793,365to Sensabaugh; U.S. Pat. No. 4,917,128 to Clearman et al.; U.S. Pat. No.4,947,974 to Brooks et al.; U.S. Pat. No. 4,961,438 to Korte; U.S. Pat.No. 4,920,990 to Lawrence et al.; U.S. Pat. No. 5,033,483 to Clearman etal.; U.S. Pat. No. 5,074,321 to Gentry et al.; U.S. Pat. No. 5,105,835to Drewett et al.; U.S. Pat. No. 5,178,167 to Riggs et al.; U.S. Pat.No. 5,183,062 to Clearman et al.; U.S. Pat. No. 5,211,684 to Shannon etal.; U.S. Pat. No. 5,247,949 to Deevi et al.; U.S. Pat. No. 5,551,451 toRiggs et al.; U.S. Pat. No. 5,285,798 to Banerjee et al.; U.S. Pat. No.5,593,792 to Farrier et al.; U.S. Pat. No. 5,595,577 to Bensalem et al.;U.S. Pat. No. 5,816,263 to Counts et al.; U.S. Pat. No. 5,819,751 toBarnes et al.; U.S. Pat. No. 6,095,153 to Beven et al.; U.S. Pat. No.6,311,694 to Nichols et al.; and U.S. Pat. No. 6,367,481 to Nichols, etal.; US Pat. Appl. Pub. No. 2008/0092912 to Robinson et al.; and PCT WO97/48294 and PCT WO 98/16125. See, also, those types of commerciallymarketed cigarettes described Chemical and Biological Studies on NewCigarette Prototypes that Heat Instead of Burn Tobacco, R.J. ReynoldsTobacco Company Monograph (1988) and Inhalation Toxicology, 12:5, p.1-58 (2000).

The extract or isolate described herein can be incorporated intosmokeless tobacco products, such as loose moist snuff, loose dry snuff,chewing tobacco, pelletized tobacco pieces (e.g., having the shapes ofpills, tablets, spheres, coins, beads, obloids or beans), extruded orformed tobacco strips, pieces, rods, cylinders or sticks, finely dividedground powders, finely divided or milled agglomerates of powdered piecesand components, flake-like pieces, molded processed tobacco pieces,pieces of tobacco-containing gum, rolls of tape-like films, readilywater-dissolvable or water-dispersible films or strips (e.g., US Pat.App. Pub. No. 2006/0198873 to Chan et al.), or capsule-like materialspossessing an outer shell (e.g., a pliable or hard outer shell that canbe clear, colorless, translucent or highly colored in nature) and aninner region possessing tobacco or tobacco flavor (e.g., a Newtoniamfluid or a thixotropic fluid incorporating tobacco of some form).Various types of smokeless tobacco products are set forth in U.S. Pat.No. 1,376,586 to Schwartz; U.S. Pat. No. 3,696,917 to Levi; U.S. Pat.No. 4,513,756 to Pittman et al.; U.S. Pat. No. 4,528,993 to Sensabaugh,Jr. et al.; U.S. Pat. No. 4,624,269 to Story et al.; U.S. Pat. No.4,987,907 to Townsend; U.S. Pat. No. 5,092,352 to Sprinkle, III et al.;and U.S. Pat. No. 5,387,416 to White et al.; US Pat. App. Pub. Nos.2005/0244521 to Strickland et al. and 2008/0196730 to Engstrom et al.;PCT WO 04/095959 to Arnarp et al.; PCT WO 05/063060 to Atchley et al.;PCT WO 05/016036 to Bjorkholm; and PCT WO 05/041699 to Quinter et al.,each of which is incorporated herein by reference. See also, the typesof smokeless tobacco formulations, ingredients, and processingmethodologies set forth in U.S. Pat. No. 6,953,040 to Atchley et al. andU.S. Pat. No. 7,032,601 to Atchley et al.; US Pat. Appl. Pub. Nos.2002/0162562 to Williams; 2002/0162563 to Williams; 2003/0070687 toAtchley et al.; 2004/0020503 to Williams, 2005/0178398 to Breslin etal.; 2006/0191548 to Strickland et al.; 2007/0062549 to Holton, Jr. etal.; 2007/0186941 to Holton, Jr. et al.; 2007/0186942 to Strickland etal.; 2008/0029110 to Dube et al.; 2008/0029116 to Robinson et al.;2008/0029117 to Mua et al.; 2008/0173317 to Robinson et al.; and2008/0209586 to Neilsen et al., each of which is incorporated herein byreference.

The amount of extract or isolate added to a tobacco composition, orotherwise incorporated within a tobacco composition or tobacco product,can depend on the desired function of that extract or isolate, thechemical makeup of that extract or isolate, and the type of tobaccocomposition to which the extract or isolate is added. The amount addedto a tobacco composition can vary, but will typically range from about 5ppm to about 5 weight percent based on the total dry weight of thetobacco composition to which the extract or isolate is added. The amountadded may vary, depending, for example, on the goal to be achieved byaddition of such compound or mixture of compounds (e.g., the enhancementof flavor) and on the specific characteristics of the compound ormixture of compounds to be added.

EXPERIMENTAL

Aspects of the present invention is more fully illustrated by thefollowing examples, which are set forth to illustrate certain aspects ofthe present invention and are not to be construed as limiting thereof.

Example 1

Extraction and Isolation of Sucrose Esters, Lutein, Cembratrienediolsand Cis-Abienol

Extraction

Nicotiana tabacum Galpao tobacco is harvested, chopped into pieces, andextracted with methanol. The leaves are removed and the methanol extractis concentrated to approximately 77% solids by weight. The concentratedmethanol extract separates into two layers, comprising an upper tar-likelayer (which is soluble in methanol) and a lower aqueous layer (which issoluble in water). The upper tar-like layer is separated and is found tocontain leaf surface analytes of interest such as sucrose esters,cembratrienediols, and cis-abienol. These analytes of interest can beseparated and collected by liquid chromatography and/or flashchromatography.

Isolation Methods

Preparative scale liquid chromatography is commonly used to separate andcollect compounds of interest in a complex mixture (e.g., using a Watersprep LC system comprising a Waters 2707 Autosampler, a Waters DADdetector, a Waters 2545 Quaternary Gradient Module, and a WatersFraction Collector III and equipped with a Waters μBondapak C18 19×300mm column and fraction collector).

Separation methods are typically developed on an analytical scale liquidchromatography column (e.g., using a Waters 2695 LC system equipped witha Waters μBondapak C18 3.9×300 mm 10 μm particle column) and scaled upto a preparative scale liquid chromatography system. Flashchromatography (e.g., using a Teledyne Isco CombiFlash Automated FlashPurification System with 275 g C18 Gold column) may also be used toisolate compounds of interest. This technique can be used alone or intandem with preparative chromatography to isolate and collect variouscompounds.

Isolation of Sucrose Esters

Sucrose esters are isolated from the tar-like layer by preparative scaleliquid chromatography. The tar-like mixture is dissolved in methanol(roughly 20 mg/mL) and injected (1 mL) on the Waters prep LC systemnoted above, operated at a flow rate of 36 mL/min, isocratic analysiswith 75:25 methanol: water, and a Waters DAD detector at 214 nm. Thefractions corresponding to the known retention time of sucrose esterstandards (collected between about 1.0 and about 5.6 minutes) arecollected and combined. Another major peak is noticed to elute with thesucrose esters at approximately 1.6 minutes. HR-LC-MS analysis of thesefractions shows that this peak corresponds to quercetin-3-rutinoside orrutin. The concentration of rutin in the tar-like mixture as determinedby HR-LC-MS analysis is approximately 800 μg/g.

To generate an extract free of the rutinoside, a liquid-liquidextraction is performed on the tar-like layer prior to prep LC. Thetar-like layer (about 2 g) is dissolved in methanol (30 mL). Thismixture is added to a reparatory funnel with distilled, deionized water(80 mL) and methylene chloride (40 mL). The mixture is shaken, theaqueous layer (shown to contain the rutin) is discarded, and themethylene chloride layer is removed and concentrated. The resultingmaterial (reconstituted in methanol) is analyzed by HR-LC-MS, whichshowed the presence of sucrose esters but not the rutinoside. Therutinoside-free material is then injected onto the prep LC system toisolate the sucrose esters.

The fractions corresponding to the known retention time of sucrose esterstandards are collected and combined. The combined fractions areconcentrated to remove methanol and methylene chloride is added to theremaining water layer in a 1:1 ratio. The tubes are shaken, centrifuged,and the aqueous layer is discarded. The methylene chloride layer isconcentrated and dissolved in isopropyl alcohol for HR-LC-MS analysis.The tar-like layer of tobacco methanol extract is determined to containapproximately 1,000 μg/g of a range of sucrose esters. This extractexhibits a similar qualitative distribution of sucrose esters as ispresent in other Oriental, cured tobacco types.

Isolation of Lutein

Lutein is isolated by prep LC from the tar-like layer, which is firsttreated to remove rutinoside, as described above. The rutinoside-freemixture is injected onto the prep LC system (10 mL injection volume)comprising a Symmetry Prep C18 19×300 mm 7 μm particle column at ambienttemperature, operated at a flow rate of 26 mL/min, a solvent system ofmethanol and water, with initial ratio of 75:25, ratio at 10 minutes of75:25, ratio at 15 minutes of 100:0, and ratio at 25 minutes of 75:25:water, and a Waters DAD detector at 443 nm. The fraction collector isset to collect 40 second fractions throughout the run, with totalanalysis time of 30 minutes. Lutein elutes at approximately 18.7 minutesunder these conditions (as correlated with a lutein standard previouslyinjected onto the prep LC system).

Lutein is also isolated from the tar-like layer by flash chromatography.A sample of the tar-like layer, which is first treated to removerutinoside, as described above, is injected (15 mL injection volume)onto the flash chromatography system. The flash chromatography system isoperated at a flow rate of 150 mL/min with a solvent (methanol andwater) gradient with initial ratio of 75:25, ratio at 5 minutes of75:25, ratio at 7 minutes of 100:0, and ratio at 15 minutes of 75:25,and a detector set at 443 nm. The fractions giving rise to a signal at443 nm after the elution of cis-abienol were collected on the flashchromatography system, combined, and concentrated (e.g., using a BuchiRotavapor unit set at 50° C. and a vacuum of 337 mBar). The combined,concentrated fractions are dissolved and injected onto the prep LCsystem (10 mL injection volume) comprising a Symmetry Prep C18 19×300 mm7 μm particle column at ambient temperature, operated at a flow rate of26 mL/min, a solvent system of methanol and water, with initial ratio of75:25, ratio at 3 minutes of 75:25, ratio at 5 minutes of 100:0, andratio at 12 minutes of 75:25: water, and a Waters DAD detector at 443nm. The fraction collector is set to collect 20 second fractionsthroughout the run, with total analysis time of 15 minutes. Luteinelutes at 9 minutes.

Lutein-containing fractions that have been isolated by prep LC and/orflash chromatography are combined and concentrated to remove methanol(e.g., using a Buchi Rotovapor unit set at 50° C. and a vacuum of 337mBar). The resulting material is a semi-solid form of lutein.

Isolation of Cembratrienediols

Cembratrienediols are isolated from the tar-like layer by preparativescale liquid chromatography. A sample of the tar-like layer describedabove, is injected (1 mL injection volume) onto the flash chromatographysystem. The flash chromatography system is operated at a flow rate of 36mL/min with an isocratic solvent system (75:25 methanol:water) and adetector set at 214 nm. The fractions giving rise to a signal at 214 nmwere collected on the flash chromatography system. The fractioncollector is set to collect 20 second fractions throughout the run, withtotal analysis time of 10 minutes. Using the retention time ofβ-cembratrienediol and α-cembratrienediol standards, the fractionscorresponding to each of these compounds were collected, and separatelycombined. On this system, the β-cembratrienediol is eluted at 9.0minutes and the α-cembratrienediol is eluted at 6.9 minutes.

The isolated fractions are separately concentrated to remove methanolfrom the fractions, leaving aqueous solutions. Methylene chloride isadded to each isolated fraction, the fractions are shaken to isolate thedesired compound in the methylene chloride layer, and the fractions arecentrifuged. The aqueous layer is discarded and the remaining methylenechloride layers are analyzed by GC/MS (e.g., using an Agilent 6890/5973system from Agilent). GC/MS is conducted by adding DMF with internalstandard (400 ppm tert-butyl hydroquinone) and BSTFA with 1% TMCS tovials containing the isolated fractions. The vials are kept at 76° C.for 30 minutes and cooled to room temperature for 30 minutes. Theresulting solutions are analyzed by GC/MS, and peak identification isdone by comparing the spectra of the silylated cembratrienediols withthe spectra of known standards.

The mass spectra confirms that the combined fractions are the α and βcembratriene diols, and the data indicates that the bothcembratrienediol fractions are approximately 99% pure based on the totalarea count. The β-cembratrienediol fraction contains a small amount ofsugar-like compounds and hexadecanoic acid and the α-cembratrienediolfraction contains small amounts of sugar-like compounds and aphytol-like compound. The tar-like layer of tobacco methanol extract isdetermined to contain approximately 80 mg/g β-cembratrienediol andapproximately 30 mg/g α-cembratrienediol.

Isolation of Cis-Abienol

Cis-abienol is isolated from the tar-like layer by preparative scaleliquid chromatography. A sample of the tar-like layer is first treatedto remove rutinoside, as described above. The rutinoside-free mixture isinjected onto the prep LC system (10 mL injection volume) comprising aSymmetry Prep C18 19×300 mm 10 μm particle column at ambienttemperature, operated at a flow rate of 36 mL/min, an isocratic solventsystem of methanol and water, with ratio of 75:25, and a Waters DADdetector at 214 nm. The fraction collector is set to collect 20 secondfractions throughout the run (of 12 mL each), with total analysis timeof 20 minutes. Cis-abienol elutes at approximately 17.0 minutes underthese conditions.

Initially, the isolated fractions with a significant absorption peak at214 nm, are unknown. Thus, the combined fractions are analyzed by GC/MS,which is inconclusive. The isolated fraction is infused onto the ThermoTSQ Quantum Ultra MS/MS using an Atmospheric Pressure ChemicalIonization probe for ionization of the sample. This technique indicatesthat the molecular weight of the compound in the isolated fraction is 29amu. The isolated fraction is additionally analyzed by UV/Visspectroscopy (e.g., using a Hewlett Packard 8453 UV/Visspectrophotometer), which shows a maximum absorption at 238 nm. Furtherconfirmation of the identity of this isolated fraction is based onreported HPLC-ESI-MS molecular weight, NMR, and UV/Vis data oncis-abienol in an article by Ding et al., Chromatographia 66:529-532(2007), which is incorporated herein by reference.

Cis-abienol is alternatively isolated from the tar-like layer by flashchromatography. A sample of the tar-like layer, which is first treatedto remove rutinoside, as described above, is injected (15 mL injectionvolume) onto the flash chromatography system. The flash chromatographysystem is operated at a flow rate of 150 mL/min with a solvent (methanoland water) gradient with initial ratio of 75:25, ratio at 5 minutes of75:25, ratio at 7 minutes of 100:0, and ratio at 15 minutes of 75:25,and a detector set at 214 nm. The fractions giving rise to a signal at214 nm are collected on the flash chromatography system, combined, andconcentrated (e.g., using a Buchi Rotavapor unit set at 50° C. and avacuum of 337 mBar). The combined, concentrated fractions are dissolvedand injected onto the prep LC system (10 mL injection volume) comprisinga Symmetry Prep C18 19×300 mm 7 μm particle column at ambienttemperature, operated at a flow rate of 26 mL/min, a solvent system ofmethanol and water, with initial ratio of 75:25, ratio at 10 minutes of75:25, ratio at 15 minutes of 100:0, and ratio at 20 minutes of 75:25,and a Waters DAD detector at 214 nm. The fraction collector is set tocollect 20 second fractions throughout the run (12 mL volume of each),with total analysis time of 30 minutes. Cis-abienol elutes at 17.8minutes.

The cis-abienol fractions collected from the preparative LC and/or theflash chromatography method are concentrated to provide a semi-solidform of cis-abienol. High volume isolation and collection of cis-abienolare typically conducted on the flash chromatography system, which allowsfor greater efficiency, as more concentrated samples can be injected. Insome cases, fractions from flash chromatography must be further resolvedusing preparative HPLC; however, flash chromatography typically providessufficient resolution of cis-abienol without the need for preparativeHPLC of the fractions.

Example 2 Extraction and Isolation of Sucrose Esters, Lutein,Cembratrienediols and Cis-Abienol

A mixture of flue-cured, burley, and Oriental tobaccos is subjected to adry steam distillation. Specifically, a strip blend of flue cured,burley, and Oriental tobaccos is placed in a 640 ft³ wagon equipped withsteam distillation capability Anhydrous steam is passed through thewagon and condensed, producing approximately 4 gallons per minute ofsteam distillate. The distillate is processed employing equipmenttraditionally employed for the isolation of peppermint oils. A fewminutes after beginning the steam distillation process, an oil sheenbegins to appear on the surface of the collected distillate. As timeprogresses, the sheen becomes a defined oil layer resting on top of thewater condensate. This reddish-brown essential oil is gently removedfrom the water.

The essential oil is dissolved in methylene chloride and analyzed by gaschromatography/mass spectrometry (GC/MS, e.g., Agilent 6890 GC equippedwith Agilent 5973 MSD). Data indicates that the essential oil comprises,as major volatile and semi-volatile compounds, solanone, neophytadiene,palmitic acid, and oleic acid. Other components of the essential oil aremegastigmatrienone isomers, ionol derivatives, β-damascenone, andnorsolanadione.

Further, the distillate water that was first passed through theessential oil isolation equipment and subsequently exhausted to thesewer (i.e., the “waste” stream) is captured. Samples of the “waste”stream collected during the distillation as a function of time aredissolved in methylene chloride and analyzed by GC/MS. The resultingchromatograms indicate that the “waste” stream contains a notable numberof volatile and semi-volatile compounds including some of the compoundsfound in the essential oil (e.g., neophytadiene, nicotine, furfurylalcohol, and bipyridine).

The “waste” stream is separated into aqueous and organic components tofacilitate downstream processing/separation of the essential oil intoless complex mixtures or individual components. Specifically, “waste”water from the steam distillation process is added to silica containedwithin a fritted glass cylinder. The water is gently removed from thesilica using a water aspirator vacuum. Hexane is percolated through thecolumn, followed by MTBE, followed by methanol. The organic solvents areremoved by rotary evaporation and the resulting materials arereconstituted in methylene chloride. The methylene chloride samples areanalyzed by GC/MS. The major component of the hexane extract isneophytadiene.

Example 3 Extraction and Isolation of Sucrose Esters andCembratrienediols

A tobacco leaf is dipped into methylene chloride at room temperature forapproximately 30 seconds. The resulting methylene chloride solution isconcentrated and the extract is cleaned using a method described inAshraf-Khorassani et al., Beitrage Tabkforsch. Int. 23: 32-45 (2008),which is incorporated herein by reference in its entirety.

The cleaned extract is purified with normal phase HPLC with UV detectionat 214 nm, using a cyano-bonded silica column (25 cm×10 mm, d_(p)=5 μm)and a mobile phase of ethanol/iso-octane/water in a ratio of 15:85:0.1.Peaks are identified for sucrose esters, α-cembrene diol and β-cembrenediol; fractions corresponding to each peak are collected individually.The identities of the fractions corresponding to each peak are confirmedby GC-FID (MS), DB5, 15 m×0.25 mm (TMS derivatives) with an initial oventemperature of 80° C. (held for 2 minutes), which was ramped up 10°C./min to 140° C., ramped up 5° C./min to 300° C. and held at 300° C.for 10 minutes.

Many modifications and other embodiments of the invention will come tomind to one skilled in the art to which this invention pertains havingthe benefit of the teachings presented in the foregoing description.Therefore, it is to be understood that the invention is not to belimited to the specific embodiments disclosed and that modifications andother embodiments are intended to be included within the scope of theappended claims. Although specific terms are employed herein, they areused in a generic and descriptive sense only and not for purposes oflimitation.

What is claimed:
 1. A method of extracting and isolating compounds fromplants of the Nicotiana species, comprising: receiving a plant materialof the Nicotiana species; contacting the plant material with a solventfor a time and under conditions sufficient to extract one or moredesired compounds from the plant material into the solvent; separatingthe solvent containing the one or more desired compounds from theextracted plant material; and purifying the solvent containing the oneor more desired compounds to provide an isolate comprising at leastabout 75 percent by weight of the one or more desired compounds, the oneor more desired compounds being selected from the group consisting ofsolanone, neophytadiene, megastigmatrienone, β-damascenone,norsolanadione, cis-abienol, α-cembratrienediol, β-cembratrienediol,lutein, degradation products thereof, and mixtures thereof, wherein thesolvent is dry steam.
 2. The method of claim 1, wherein the plantmaterial of the Nicotiana species is provided in green form.
 3. Themethod of claim 1, wherein the plant material of the Nicotiana speciesis provided in cured form.
 4. The method of claim 1, wherein the plantmaterial of the Nicotiana species is in a form selected from the groupconsisting of whole leaf, laminae, cut filler, volume expanded, stems,cut-rolled stems, cut-puffed stems, reconstituted tobacco, andparticulate.
 5. The method of claim 1, wherein the one or more desiredcompounds are selected from the group consisting of solanone,neophytadiene, megastigmatrienone, β-damascenone, and norsolanadione. 6.The method of claim 1, wherein the contacting step further comprisescollecting a distillate.
 7. The method of claim 6, wherein thedistillate comprises a water layer and an oily layer.
 8. The method ofclaim 7, wherein the solvent containing the one or more desiredcompounds is selected from the group consisting of the oily layer, thewater layer, and a waste stream generated from the distillation process.9. The method of claim 1, wherein the purifying step comprises usingpreparative scale liquid chromatography.
 10. The method of claim 1,wherein the purifying step comprises using flash chromatography.
 11. Themethod of claim 1, wherein the purifying step provides an isolatecomprising greater than about 90% by weight of the one or more desiredcompounds.
 12. The method of claim 1, wherein the purifying stepprovides an isolate comprising greater than about 95% by weight of theone or more desired compounds.
 13. The method of claim 1, furthercomprising adding the isolate to a tobacco composition adapted for usein a smoking article or a smokeless tobacco composition.
 14. The methodof claim 1, further comprising treating the isolate to provide one ormore degradation products therefrom, wherein the treating is selectedfrom oxidation, heat treatment, and a combination thereof.
 15. A methodof extracting and isolating compounds from plants of the Nicotianaspecies, comprising: receiving a plant material of the Nicotianaspecies; contacting the plant material with a solvent for a time andunder conditions sufficient to extract one or more desired compoundsfrom the plant material into the solvent; separating the solventcontaining the one or more desired compounds from the extracted plantmaterial; purifying the solvent containing the one or more desiredcompounds to provide an isolate comprising at least about 75 percent byweight of the one or more desired compounds, the one or more desiredcompounds being selected from the group consisting of solanone,neophytadiene, megastigmatrienone, β-damascenone, norsolanadione,cis-abienol, α-cembratrienediol, β-cembratrienediol, lutein, degradationproducts thereof, and mixtures thereof; and treating the isolate toprovide one or more degradation products therefrom, wherein the treatingis selected from oxidation, heat treatment, and a combination thereof,wherein the isolate comprises lutein and wherein the one or moredegradation products are selected from the group consisting ofmegastigmatrienone, beta-damascenone, and mixtures thereof, or whereinthe isolate comprises cis-abienol and wherein the one or moredegradation products are selected from the group consisting ofsclareolide, sclareol, ambroxide, and mixtures thereof.
 16. The methodof claim 15, wherein the plant material of the Nicotiana species is in aform selected from the group consisting of whole leaf, laminae, cutfiller, volume expanded, stems, cut-rolled stems, cut-puffed stems,reconstituted tobacco, and particulate.
 17. The method of claim 15,wherein the plant material of the Nicotiana species is provided in greenform.
 18. The method of claim 15, wherein the plant material of theNicotiana species is provided in cured form.
 19. The method of claim 15,wherein the solvent is methanol.
 20. The method of claim 15, wherein thepurifying step comprises using preparative scale liquid chromatography.21. The method of claim 15, wherein the purifying step comprises usingflash chromatography.
 22. The method of claim 15, wherein the purifyingstep provides an isolate comprising greater than about 90% by weight ofthe one or more desired compounds.
 23. The method of claim 15, whereinthe purifying step provides an isolate comprising greater than about 95%by weight of the one or more desired compounds.
 24. The method of claim13, wherein the isolate is added in the form of a casing formulation ora top dressing formulation applied to tobacco strip or in the form of acomponent of a reconstituted tobacco material.
 25. The method of claim13, wherein the tobacco composition comprises a tobacco material adaptedfor use in a smoking article.
 26. The method of claim 25, wherein theamount of isolate in the tobacco composition is between about 5 ppm andabout 5 weight percent based on the total dry weight of the tobaccomaterial in the smoking article.
 27. The method of claim 13, wherein thetobacco composition comprises a tobacco material adapted for use in asmokeless tobacco product.
 28. The method of claim 27, wherein theamount of isolate in the tobacco composition is between about 5 ppm andabout 5 weight percent based on the total dry weight of the tobaccomaterial in the smokeless tobacco product.
 29. The method of claim 15,further comprising: adding the isolate to a tobacco composition adaptedfor use in a smoking article or a smokeless tobacco composition.
 30. Themethod of claim 29, wherein the isolate is added in the form of a casingformulation or a top dressing formulation applied to tobacco strip or inthe form of a component of a reconstituted tobacco material.
 31. Themethod of claim 29, wherein the tobacco composition comprises a tobaccomaterial adapted for use in a smoking article.
 32. The method of claim31, wherein the amount of isolate in the tobacco composition is betweenabout 5 ppm and about 5 weight percent based on the total dry weight ofthe tobacco material in the smoking article.
 33. The method of claim 29,wherein the tobacco composition comprises a tobacco material adapted foruse in a smokeless tobacco product.
 34. The method of claim 33, whereinthe amount of isolate in the tobacco composition is between about 5 ppmand about 5 weight percent based on the total dry weight of the tobaccomaterial in the smokeless tobacco product.